For those tissue-specific proteins that lack sufficient AIRE-dependent thymic expression to ensure central tolerance, peripheral tolerance mechanisms exist to ensure health and the avoidance of autoimmunity. In addition totheperipheral deletion ofI cells with high self Agreactivity, the clonal anergy mechanismis available to limit the proliferative potential of potentially autoreactive T cells. Furthermore, natural CD4+ regulatory T cells accumulate in the periphery and exert a counter-regulatory influence on T cell responsiveness to limit immunopathology. In the most recent grant period, we discovered that in the setting of profound lymphopenia (and in the absence of regulatory T cells) clonal anergy cannot be induced, whereas following partial immune reconstitution CD25+ CD4+ regulatory T cells facilitate the induction clonal anergy in T cells that recognize Ag in absence of infection or adjuvant. We now propose to explore in detail this inter- relationship between CD4+ regulatory T cells and the induction and/or reversal of clonal anergy in both the lymphopenic and immunocompetent immune system. Specifically, this application seeks to: 1) define intracellular signaling events that mediate a resistance to T cell clonal anergy induction and its reversal within lymphopenic individuals, 2) identify molecules that promote the development CD4+ regulatory T cells early during the recovery from lymphopenia, and 3) examine the inter-dependence of CD4+ regulatory T cell generation and the induction of T cell clonal anergy in immunocompetent individuals. Data generatedfrom this work will serve as the basis for the development of Ag-specific strategies to induce transplantation tolerance or cure clinical autoimmune disease.